Protease has for a long time been used in the industrial field and applied to an extremely wide variety of products such as detergents including fabric detergents, fiber-reforming agents, leather treatment agents, cosmetics, bath salts, food reforming agents and medicinal products. Among them, the protease for detergent use is industrially produced in the largest amount. For example, Alcalase, Savinase (registered trade mark; Novozymes), Maxacal (registered trade mark; Genencor), BLAP (registered trade mark; Henkel) and KAP (Kao Corp.) are generally known.
A purpose for adding a protease to a detergent is to decompose stain mainly constituted of proteins attached to a clothing material into low-molecular substances, thereby accelerating solubilization with a surfactant. However, stain is actually a composite which contains not only proteins but also a plurality of components including organic substances and inorganic substances (such as lipids derived from sebum and solid particles) in combination. Then, alkaline proteases of about 43,000 in molecular weight exhibiting excellent washing performance against composite stain (i.e. containing not only protein but also sebum and others) have been developed and patent applicated (see Patent Document 1). The alkaline proteases differ from subtilisin, which is a conventionally known serine protease derived from a Bacillus genus bacteria, having different molecular weight, primary structure and enzymatic properties, and particularly differ from subtilisin in that the alkaline proteases have extremely strong oxidant resistance. Accordingly, it has been proposed that the alkaline proteases are classified into a new subfamily of subtilisin (Non Patent Document 1).
Detergents can be classified into both a powder detergent and a liquid detergent based on their forms. The liquid detergent has excellent solubility compared to the powder detergent. The liquid detergent has a merit since an undiluted liquid detergent can be directly applied to stains. Also recently, a liquid detergent (so called, a concentrated liquid detergent), which functions equivalently to a conventional liquid detergent by using a half amount, has been commercially available. Since such a concentrated liquid detergent is contained in a small container, it does not require a large storage space or a large amount of fuel for transportation. In addition to these advantages, based on thorough reconsideration of washing mechanism, some products show improved rinsing performance. In this manner, time required for laundry can be reduced and water required can be saved.
In order to constantly maintain a predetermined enzymatic activity of an enzyme-containing liquid detergent, it is necessary to stabilize the enzyme such as a protease dissolved in the liquid detergent. Furthermore, to enhance the detergency of a detergent, it is desirable to add an enzyme to the detergent as much as possible. However, it is widely known that it is technically difficult to stably mix an enzyme with a liquid detergent. For example, storing an enzyme in a liquid at normal temperature easily cause denaturation of a protein. In addition, a surfactant, a fatty acid, a solvent and others are contained in a liquid detergent and the pH thereof is weak alkali. Thus, the liquid detergent is extremely severe environmental conditions for an enzyme. Furthermore, a protease is a proteolytic enzyme. Because of the feature, protease suffers from autodigestion, which makes it further difficult to stably store the protease in a liquid detergent. Moreover, low water content of the concentrated liquid detergent (concentrated compared to a conventional liquid detergent) makes it difficult to dissolve a large amount of enzyme.
As a technique for stably maintaining enzymatic activity in a liquid detergent, it is known to add an enzyme stabilizer such as a calcium ion, borax, boric acid, a boron compound, a carboxylic acid such as formic acid, and a polyol. Furthermore, overcoming autodigestion by inhibiting protease activity has been studied. Methods of stabilizing a protease by reversible inhibition of the protease by 4-substituted phenyl boronic acid (Patent Document 2) and by a certain peptide aldehyde and a boron composition (Patent Document 3) have been reported. Moreover, it is reported that a protease chemically modified with dextran can be improved in stability in an aqueous solution containing a surfactant (Non Patent Document 2). In addition, a protease mutant improved in stability to a surfactant is also known (Patent Documents 4 to 6).
In contrast, a technique for improving solubility of an enzyme to a liquid detergent has not yet been developed up to the present.